1. Volume 19, Issue 4, Pages 561-569 (October 2003)
Natural Killer Cells Activated by MHC Class ILow Targets Prime Dendritic Cells to Induce Protective CD8 T Cell Responses 
Ralph Mocikat, Heidi Braumüller, Alain Gumy, Oliver Egeter, Heike Ziegler, Uwe Reusch, Anja Bubeck, Jacques Louis, Reinhard Mailhammer, Gert Riethmüller, Ulrich Koszinowski, Martin Röcken 
Immunity 
Volume 19, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1 In Vitro Characterization of MHC Class Ilow Tumor Cells
(A) MHC class I downregulation in m152-transfected A20 cells (gray area), as detected by FITC-labeled anti-H-2Dd mAb HB87. The hatched area represents A20 wild-type tumor, the white area the isotype control. When compared to the parental cells, the remaining H2Dd mean fluorescence intensity of the transfectants used in the experiments was 12% for A20-m152, 23% for A20-m06, and 19% for CT26-m06, respectively. Cells transfected with empty cassettes had normal MHC class I expression. Reduced MHC class I expression by CMV gene-transfected cells was verified prior to all experiments.
(B) Expression of H-2 and various other surface molecules by m152-transfected A20 cells (filled bars) or by A20 wild-type tumors (open bars). Isotype controls had a mean fluorescence <8. Representative results from at least two experiments.
(C) Specific lysis of m152-transfected A20low or A20 wild-type lymphoma by TCR-transgenic influenza hemaglutinin (HA)-specific CTL. Tumors were pulsed overnight with 3H-thymidine and HA peptide, and specific lysis was determined using the JAM test as described in the Experimental Procedures. Experiments were performed in triplicates.
Immunity  , DOI: ( /S (03) )

3. Figure 2 Rejection of MHC Class Ilow Tumor Cells Requires NK Cells
(A) Survival of BALB/c mice after injection of 106 A20 or 106 CT26 cells. Injected tumor cells were either controls or cells transfected with either the m152 gene (A20low-m152) or the m06 gene (A20low-m06 or CT26low-m06). Tumor cells transfected with empty vector cassettes showed identical growth kinetics as wild-type cells.
(B) Survival of BALB/c mice or NK cell-depleted BALB/c mice after injection of A20low cells. NK cells were depleted using rabbit anti-asialo GM1 antibody.
(C) Survival of BALB/c mice after injection of m152-transfected A20 cells, following the restoration of MHC class I expression by preincubation with 670 U/ml IFN-γ.
Immunity  , DOI: ( /S (03) )

4. Figure 3
MHC Class Ilow Tumors Activate NK Cells to Prime DC for IL-12 Production and for the Induction of CD8+ T Cell Responses
(A) IFN-γ mRNA expression by sorted NK cells, DC, and non-NK cells from spleens 4 hr after injection of either A20 or A20low lymphoma cells. Some of the mice had received anti-IFN-γ mAb 4 hr before tumor challenge. The insert shows IFN-γ+ DC, NK, or T cells in the spleen detected by an IFN-γ capture assay and counterstained for CD11c, DX5, and CD3 expression. The high amounts of IFN-γ detected in mice treated with A20low and anti-IFN-γ are reproducible and are due to the enrichment of IFN-γ on the cell surface after anti-IFN-γ mAb treatment.
(B) IL-12p40 mRNA expression by sorted CD11c+ DC or non-DC from spleens 4 hr after intravenous injection of either A20 or A20low lymphoma cells. Some of the mice had received anti-IFN-γ antibody 4 hr before tumor challenge.
(C) IFN-γ mRNA expression in CD3+CD8+ T cells 4 days after injection of either A20 or A20low lymphoma cells. Some of the mice received anti-IFN-γ mAb 4 days before injection of A20low lymphoma cells. The insert shows percentages of IFN-γ+ DC, NK, or T cells in the spleen detected by an IFN-γ capture assay and counterstained for CD11c, DX5, and CD3 expression. All panels show one out of two similar experiments.
Immunity  , DOI: ( /S (03) )

5. Figure 4 T Cell Priming Is Dependent on NK Cells and IL-12
(A) IFN-γ and HPRT expression by NK cells (upper two lanes) or IL-12p40 and HPRT expression by DC (lower two lanes). Mice received either A20 lymphoma or A20low lymphoma cells. In half of the mice, NK cells were depleted 3 days before by rabbit anti-asialo GM1 antibody. Four hours after intravenous tumor cell injection, the indicated cell population was positively sorted and mRNA was amplified and determined as described (Biedermann et al., 2001). N.D., not detectable because of efficient NK cell depletion (confirmed by FACS analysis).
(B) Four days after injection of either A20 or A20low lymphoma cells, CD3+CD8+ T cells were isolated and IFN-γ mRNA expression was determined by quantitative RT-PCR. In half of the animals, NK cells were depleted.
(C) Percentages of IFN-γ+CD3+ spleen cells as determined by an IFN-γ capture assay after treating mice either with A20, A20low, or A20low + anti-IL-12 mAb.
Immunity  , DOI: ( /S (03) )

6. Figure 5
DC-Activation by MHC Class Ilow Tumors Is T Cell Independent, but Rejection of MHC Class Ilow Tumor Cells Is T Cell Dependent
(A) Percentages of IL-12-producing DC as determined by intracellular FACS staining of spleen cells from SCID mice after treatment with either A20, A20low, or A20low + anti-IFN-γ mAb.
(B) Survival of BALB/c nude/nude mice injected with either A20 wild-type cells or m152-transfected A20 MHC class Ilow cells.
(C) Survival of SCID mice after injection of either 106 m152-transfected A20 MHC class Ilow cells or 106 A20 wild-type cells. Half of the SCID mice had received 10 nMol CpG-DNA 1668 oligonucleotide on days –1 and 1 of the tumor cell transfer.
Immunity  , DOI: ( /S (03) )

7. Figure 6
NK Cell-Induced Activation of DC Establishes Long-Lasting Memory
(A) Long-term memory induced by NK cell-mediated rejection of A20low variants. BALB/c mice were primed with either 105 or 106 of MHC class Ilow A20 cells or remained unprimed. A fourth group of SCID mice was primed with 10 nMol CpG-DNA and106 MHC class Ilow A20 cells. On day +100, mice received 106 A20 wild-type tumor cells and survival was monitored.
(B) Rejection of A20low lymphoma cells in BALB/c mice requires NK and CD8+ T cells. BALB/c mice were depleted of the indicated cell populations with mAb (CD8 cells with RmCD8-2, CD4 cells with RmCD4-2, NK cells with anti-IL-2 receptor β chain TM-β1) 3 days prior to the injection of 106 A20low lymphoma cells.
Immunity  , DOI: ( /S (03) )

8. Figure 7
Rejection of MHC Class Ilow A20 Lymphoma Cells Establishes T Cell Memory Responses
(A) Four days after injection of A20low lymphoma, A20-specific T cell lines were generated. After 10 days, these T cell lines were stimulated with either APC alone or with APC and irradiated A20 cells. No T cell line could be established from NK cell-depleted mice. IFN-γ production was determined in the supernatant by ELISA. Representative data from three independent experiments which yielded similar results.
(B) Long-term memory induced by NK cell-mediated rejection of A20low variants depends on T cells. BALB/c mice were primed with 106 of MHC class Ilow A20 cells and, on day 100, re-challenged with 106 A20 wild-type tumor cells. In some of the mice, total T cells were depleted 3 days prior to the second challenge with mAb MmT1. The data are part of the experiment shown in Figure 6A.
Immunity  , DOI: ( /S (03) )